BIOLOGY 479 INTEGRATED PHYSIOLOGY LABORATORY Dept. of Biological & Allied Health Sciences BLOOMSBURG UNIVERSITY of PENNSYLVANIA.

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1 BIOLOGY 479 INTEGRATED PHYSIOLOGY LABORATORY Dept. of Biological & Allied Health Sciences BLOOMSBURG UNIVERSITY of PENNSYLVANIA LAB REPORT: Investigating Aerobic Respiration Michael Tekin Lab section: 02A, Tuesday, Experiment date: 04 March 2014 Report submitted: 11 March 2014

2 Apriori Predictions Table 1: Predictions of how cellular respiration will be affected by each combination of substrate and inhibitor. Substrate Ascorbate Malate Succinate Complex I inhibitor unaffected decreased unaffected Complex II inhibitor unaffected unaffected decreased Complex III inhibitor unaffected decreased decreased Complex IV inhibitor decreased decreased decreased Experimental Design. First the rate of oxygen consumption was determined for the mitochondria without any substrate. Since no nutrients were available, no cellular respiration should occur (Surmacz et al. 2013). Then, Succinate was. Succinate was before the inhibitor to determine whether or not the electron transport chain (ETC) was compromised by the extraction process. Thus, if the extraction process was successful, there should be an observable rate of oxygen consumption. Next, the inhibitor was. Succinate donates electron to FAD forming FADH2. FAD/FADH2 is part of complex II (Surmacz et al. 2013). The electrons than travels through the ETC in the following order: Ubiquinone, complex III, cytochrome C, Complex IV, and oxygen. Therefore, a reduction in cellular respiration would indicate that Complexes II, III, or IV was inhibited. If complex I was inhibited cellular respiration would not be affected. Then Ascorbate was. Ascorbate passes electrons directly to cytochrome C (Surmacz et al. 2013). The electrons then travel to complex IV and then oxygen. Therefore, when Ascorbate is, the metabolic rate will increase unless complex IV is inhibited. Then, another trial was run. First Malate was, followed by an inhibitor. Malate is oxidized into Oxaloacetate by NAD+ (Surmacz et al. 2013). The NAD+ takes electrons from Malate making NADH. Then NADH donates electron to complex I. Complex I then passes ETC

3 in the following order: Ubiquinone, complex III, cytochrome C, Complex IV, and oxygen. If the inhibitor caused a reduction in cellular respiration, and thus oxygen consumption, then complex I, III, or IV must be affected. If the inhibitor interferes with Complex II, cellular respiration would not be affect. When each of these substrates and inhibitor were the change in oxygen consumption was recorded and compared to predicted changes in metabolism (table 1). By comparing the observed changes in oxygen consumption, and thus cellular respiration to the predicted changes in cellular respiration the complex the inhibitor was affecting was determined. Results Recording Time (min) Figure 1. Change of oxygen after each substrate and inhibitor was. The data collected was compressed and labeled by trial and when the inhibitor or substrate was. The Mitochondria and Inhibitor treatment both show a slight increase in oxygen levels, while the Succinate, and Ascorbate showed a decrease in

4 oxygen. In Trial 2, the Malate treatment caused a decrease in oxygen levels, while the inhibitor treatment experienced no change. These results indicate the inhibitor affected complex III. Table 2: Observed oxygen consumption as substrates are Order Oxygen consumption Percent inhibition substrates were (nmole/l/min) trial 1 Mitochondria 310 N/A Succinate 4300 N/A Inhibitor A 140% B Ascrobate No inhibition Trial 2 Malate 7700 N/A inhibitor 0.00 %100 A. Oxygen appeared to be produced in this treatment. This was most likely due to oxygen diffusing into the sample due to an incomplete seal of the mitochondria chamber. B. increase in oxygen levels cause a value greater than 100%. This can be assumed to be 100% inhibition. Sample calculation S = substrate oxygen consumption SI = substrate and inhibitor oxygen consumption M = mitochondria oxygen consumption S SI M X 100 S M Example: Malate 7700nmole/L/min 0nmole/L/min 310nmole/L/min 7700 nmole/l/min 310nmole/L/min X 100 = 100% Observation In the first trial, mitochondria alone had a 310 nmole/l/min rate of oxygen consumption. When Succinate was add oxygen consumption was more pronounced at 4300 nmole/l/min. Then the inhibitor was and oxygen consumption was observed at nmole/l/min. This was a

5 140% inhibition. Last, Ascorbate was oxygen consumption resumed at a rate of nmole/l/min (Table 2). In the second trial Malate was. The mitochondria consumed at a rate of 7700 nmole/l/min. When the inhibitor was and the mitochondria experienced 100% inhibition and oxygen consumption was 0 nmole/l/min (table 2). Discusion and conclusion Since there was low levels of oxygen loss with no substrate it can be assumed mitochondria alone does not cause a drastic decrease in oxygen. Since Succinate caused a large decrease in rate of oxygen loss the ETC appeared to be intact. The following changes in oxygen levels were observed when the following subsrates interacted with the inhibited mitochondria: Succinate caused a increase in oxygen level, Malate caused no change in oxygen levels, and acerbate caused a decrease in oxygen levels (figure 1). Moreover, Succinate experienced a 140% inhibition and Malate experienced 100% inhibition, while Ascorbate was not inhibited This trend resembles the expected pattern for complex III inhibition (Table 1.) The only exception is Succinate and inhibitor caused a slight decrease in oxygen. This is most likely due to oxygen diffusing into the solution from an incomplete seal of the mitochondria chamber since mitochondria have no mechanism for producing oxygen (Surmacz et al. 2013). Questions 1. What would happen to oxygen consumption if you succinate to a mitochondrial suspension poisoned with rotenone? Explain.

6 Rotenone binds to complex I (Mckee et al. 2009). Succinate donates electron to the ETC via FAD on complex II. The electrons then go to the Ubiquinone pool and onwards through the ETC (Surmacz et al. 2013). Therefore, since complex I is not involved with this particular metabolic pathway, oxygen consumption will not decrease. 2. After exhaustive experimentation, you cannot find a single substrate capable of stimulating oxygen consumption in a mitochondrial suspension poisoned with KCN. Where is the site of inhibition? Explain. The Cyanide ion irreversibly binds to complex IV preventing oxygen from being the final electron acceptor (Mckee et al. 2009). Therefore, the ETC becomes backed up and cellular respiration, and thus O2 consumption halts (Surmacz et al. 2013). Since complex IV is at the end of the ETC, no substrate can bypass it. Application: 1. Dinitrophenol is considered to be an uncoupling reagent. Uncoupling reagents dissociate ATP synthesis from electron transport in mitochondria, allowing electron transport to occur in the absence of ATP synthesis. DNP is highly toxic to humans. For a brief period in the 1940's, however, sub-lethal doses of DNP were prescribed as a means of weight reduction humans. A. Why would DNP be used as a means of weight reduction? Uncoupling reagents decrease the proton gradient (Mckee et al. 2009). This decreases the rate of cellular respiration and overall metabolic efficiency of the mitochondria. Moreover, the

7 decrease rate of cellular respiration causes ADP levels to increase. This stimulates Lipolysis and beta oxidation. Therefore, fat is removed from the adipose tissue causing weight loss. B. In many vertebrates brown-adipose tissue is a heat-producing tissue. How does it produce heat? Brown adipose has uncoupled mitochondria (Mckee et al. 2009). Uncoupled mitochondria are mitochondria with uncouplers present in them. These uncoupling reagents reduce the proton gradient by transporting hydrogen ions across the membrane. The decrease in potential energy is then released as heat. Regular tissues have coupled mitochondria or mitochondria without uncouplers. These mitochondria do not produce heat. Literature Cited McKee T and Mckee J R Biochemistry:the Molecular Basis of Live. 5th ed. Oxford University Press Surmacz C and Coleman W Investigating Aerobic Respiration. Lab Manual for Integrated Physiology Laboratory. Bloomsburg University of Pennsylvania, Bloomsburg, PA. p